The present invention generally relates to compositions and methods for preventing and treating human diseases including, but not limited to, pathogens such as bacteria, yeast, parasites, fungus, viruses, and cancer. More specifically, embodiments described herein concern the manufacture and use of ligand/receptor specificity exchangers, which redirect existing antibodies in a subject to receptors present on pathogens.
Infection by pathogens, such as bacteria, yeast, parasites, fungus, and viruses, and the onset and spread of cancer present serious health concerns for all animals, including humans, farm livestock, and household pets. These health threats are exacerbated by the rise of strains that are resistant to vaccination and/or treatment. In the past, practitioners of pharmacology have relied on traditional methods of drug discovery to generate safe and efficacious compounds for the treatment of these diseases. Traditional drug discovery methods typically involve blindly testing potential drug candidate-molecules, often selected at random, in the hope that one might prove to be an effective treatment for some disease. With the advent of molecular biology, however, the focus of drug discovery has shifted to the identification of molecular targets associated with the etiological agent and the design of compounds that interact with these molecular targets.
One promising class of molecular targets are the receptors found on the surface of bacteria, yeast, parasites, fungus, viruses, and cancer cells, especially receptors that allow for attachment to a host cell or host protein (e.g., an extracellular matrix protein). Research in this area primarily focuses on the identification of the receptor and its ligand and the discovery of molecules that interrupt the interaction of the ligand with the receptor and, thereby, block adhesion to the host cell or protein. Although several receptor antagnosists have promising therapeutic potential, there still remains a need for new compositions and methods to treat and prevent infection by pathogens and other diseases.
The invention described herein concerns the manufacture, characterization, and use of novel agents that bind receptors on pathogens and redirect antibodies present in a subject to the pathogen. Embodiments include a ligand/receptor specificity exchanger having at least one specificity domain comprising a ligand for a receptor and at least one antigenic domain joined to said specificity domain, wherein said antigenic domain comprises an epitope of a pathogen or toxin.
Some embodiments of the ligand/receptor specificity exchanger have a specificity domain that comprises at least three consecutive amino acids of a peptide selected from the group consisting of an extracellular matrix protein, a ligand for a receptor on a virus, and a ligand for a receptor on a cancer cell. In some aspects of this embodiment, for example, the peptide is an extracellular matrix protein selected from the group consisting of fibrinogen, collagen, vitronectin, laminin, plasminogen, thrombospondin, and fibronectin. Preferably, the extracellular matrix protein comprises at least 3 amino acids of the alpha-chain of fibrinogen and in the most preferred embodiments the ligand comprises the sequence Arginine-Glycine-Aspartate (RGD).
In other embodiments, the peptide described above is a ligand for a receptor on a virus selected from the group consisting of T4 glycoprotein and hepatitis B viral envelope protein. In still other aspects of this embodiment, the peptide is a ligand for a receptor on a cancer cell selected from the group consist of a ligand for HER-2/neu and a ligand for an integrin receptor. Preferred embodiments have a specificity domain that comprises a sequence provided by one of SEQ. ID. Nos. 1-42.
The ligand/receptor specificity exchangers described herein interact with a receptor found on a pathogen. In some embodiments, the receptor is a bacterial adhesion receptor, for example, a bacterial adhesion receptor selected from the group consisting of extracellular fibrinogen binding protein (Efb), collagen binding protein, vitronectin binding protein, laminin binding protein, plasminogen binding protein, thrombospondin binding protein, clumping factor A (ClfA), clumping factor B (ClfB), fibronectin binding protein, coagulase, and extracellular adherence protein.
The ligand/receptor specificity exchangers described herein also interact with a an antibody present in a subject. In some embodiments, for example, the antigenic domain comprises at least three amino acids of a peptide selected from the group consisting of a herpes simplex virus protein, a hepatitis B virus protein, a TT virus protein, and a poliovirus protein. In desirable embodiments, the ligand/receptor specificity exchanger has an antigenic domain that is a herpes simplex virus protein comprising a sequence selected from the group consisting of SEQ. ID. No. 53 and SEQ. ID. No. 54. In other desired embodiments, the antigenic domain is a hepatitis B virus protein comprising a sequence provided by one of SEQ. ID. No. 49, SEQ. ID. No. 50, SEQ. ID. No. 52, and SEQ. ID. No. 59.
Some ligand/receptor specificity exchangers also have an antigenic domain that is a TT virus protein comprising a sequence provided by one of SEQ. ID. Nos. 43-47 and SEQ. ID. Nos. 55-58. The ligand/receptor specificity exchangers can also have an antigenic domain that is a polio virus protein comprising a sequence selected from the group consisting of SEQ. ID. No. 48 and SEQ. ID. No. 51. Preferably, the ligand/receptor specificity exchanger has an antigenic domain that interacts with a high-titer antibody. In some embodiments, for example, the antigenic domain specifically binds to an antibody present in animal serum that has been diluted to between approximately 1:100 to 1:1000 or greater. The specificity exchangers of SEQ. ID. Nos. 60-105 are embodiments of the invention.
Aspects of the invention also concern method of treating or preventing a infection or proliferation of a pathogen. One approach for example, involves a method for treating and preventing bacterial infection. This method is practiced by providing a therapeutically effective amount of a ligand/receptor specificity exchanger to a subject, wherein said ligand/receptor specificity exchanger comprises a specificity domain that has a ligand that interacts with a receptor on a bacteria, and an antigenic domain that comprises an epitope for a pathogen or toxin. A method of treating or preventing viral infection is also an embodiment. Accordingly, a method of treating or preventing a viral infection is practiced by providing a therapeutically effective amount of a ligand/receptor specificity exchanger to a subject, wherein said ligand/receptor specificity exchanger comprises a specificity domain that has a ligand that interacts with a receptor on a virus, and an antigenic domain that comprises an epitope for a pathogen or toxin. Similarly, a method of treating or preventing cancer is an embodiment and this method can be practiced by providing a therapeutically effective amount of a ligand/receptor specificity exchanger to a subject, wherein said ligand/receptor specificity exchanger comprises a specificity domain that has a ligand that interacts with a receptor on a cancer cell, and an antigenic domain that comprises an epitope for a pathogen or toxin.
The following describes the manufacture, characterization, and use of novel agents that bind receptors on pathogens and redirect antibodies present in a subject to the pathogen. The embodiments are collectively referred to as xe2x80x9cligand/receptor specificity exchangersxe2x80x9d. The term xe2x80x9cligand/receptor specificity exchangersxe2x80x9d refers a specificity exchanger that comprises a xe2x80x9cspecificity domainxe2x80x9d that has at least one ligand for a receptor (a xe2x80x9cligandxe2x80x9d is not an antibody or portion thereof) joined to an xe2x80x9cantigenic domainxe2x80x9d that has at least one epitope of a pathogen or toxin (e.g., pertussis toxin or cholera toxin).
The ligand/receptor specificity exchangers can comprise more than a specificity domain and an antigenic domain. For example, some ligand/receptor specificity exchangers comprise a plurality of specificity domains and/or antigenic domains. Ligand/receptor specificity exchangers having multiple specificity domains and/or antigenic domains are said to be xe2x80x9cmultimerizedxe2x80x9d because more than one specificity domain and/or antigenic domain are fused in tandem. Other embodiments concern ligand/receptor specificity exchangers that contain, in addition to a specificity domain and an antigenic domain, sequences that facilitate purification (e.g., a poly histidine tail), linkers (e.g., biotin and/or avidin or streptavidin or the flexible arms of xcex phage (xcex-linkers)), and sequences or modifications that either promote the stability of the ligand/receptor specificity exchanger (e.g., modifications that provide resistance to protease digestion) or promote the degradation of the ligand/receptor specificity exchanger (e.g., protease cleavage sites). Although the specificity and antigenic domains are preferably peptides; some ligand/receptor specificity exchangers have specificity and antigenic domains that are made of modified or derivatized peptides, peptidomimetics, or chemicals.
The diversity of ligand/receptor specificity exchangers is vast because the embodiments described herein can bind to many different receptors on many different pathogens. Thus, the term xe2x80x9cpathogenxe2x80x9d is used herein in a general sense to refer to an etiological agent of disease in animals including, but not limited to, bacteria, parasites, fungus, mold, viruses, and cancer cells. Similarly, the term xe2x80x9creceptorxe2x80x9d is used in a general sense to refer to a molecule (usually a peptide other than a sequence found in an antibody, but can be a carbohydrate, lipid, or nucleic acid) that interacts with a xe2x80x9cligandxe2x80x9d (usually a peptide other than a sequence found in an antibody, or a carbohydrate, lipid, nucleic acid or combination thereof). A xe2x80x9creceptorxe2x80x9d, as used herein, does not have to undergo signal transduction and can be involved in a number of molecular interactions including, but not limited to, adhesion (e.g., integrins) and molecular signaling (e.g., growth factor receptors). For example, desired specificity domains comprise a ligand that has a peptide sequence that is present in an extracellular matrix protein (e.g., fibrinogen, collagen, vitronectin, laminin, plasminogen, thrombospondin, and fibronectin) and some specificity domains comprise a ligand that interacts with a bacterial adhesion receptor (e.g., extracellular fibrinogen binding protein (Efb), collagen binding protein, vitronectin binding protein, laminin binding protein, plasminogen binding protein, thrombospondin binding protein, clumping factor A (ClfA), clumping factor B (ClfB), fibronectin binding protein, coagulase, and extracellular adherence protein).
In other embodiments, the specificity domain comprises a ligand that has a peptide sequence that interacts with a viral receptor (e.g., a fragment of T4 glycoprotein that binds gp120 or a fragment of the preS domain, which binds gp170 of the hepadnavirus family). In still other embodiments, the specificity domain comprises a ligand that interacts with a receptor on a cancer cell (e.g., HER-2/neu (C-erbB2)) or an integrin receptor such as a vitronectin receptor, a laminin receptor, a fibronectin receptor, a collagen receptor, a fibrinogen receptor, an xcex14xcex21 receptor, an xcex16xcex21 receptor, an xcex13xcex21 receptor, an xcex15xcex21 receptor, and an xcex1vxcex23 receptor. Preferred embodiments, however, have a specificity domain that comprises at least 8 amino acids of the alpha-chain of fibrinogen and/or the sequence Arginine-Glycine-Aspartic acid (RGD) and the most preferred embodiments have a specificity domain that comprises a sequence selected from the group consisting of SEQ. ID. Nos. 60-105.
Desired antigenic domains have an epitope that is recognized by an antibody that already exists in a subject. For example, many people are immunized against childhood diseases including, but not limited to, small pox, measles, mumps, rubella, and polio. Thus, antibodies to epitopes on these pathogens can be produced by an immunized person. Desirable antigenic domains have an epitope that is found on one of these etiological agents.
Some embodiments have antigenic domains that interact with an antibody that has been administered to the subject. For example, an antibody that interacts with an antigenic domain on a ligand/receptor specificity exchanger can be co-administered with the ligand/receptor specificity exchanger. Further, an antibody that interacts with a ligand/receptor specificity exchanger may not normally exist in a subject but the subject has acquired the antibody by introduction of a biologic material (e.g., serum, blood, or tissue). For example, subjects that undergo blood transfusion acquire numerous antibodies, some of which can interact with an antigenic domain of a ligand/receptor specificity exchanger.
The most desirable antigenic domains comprise an epitope that is recognized by a high titer antibody. By xe2x80x9chigh titer antibodyxe2x80x9d is meant an antibody that has high affinity for an antigen (e.g., an epitope on an antigenic domain). For example, in a solid-phase enzyme linked immunosorbent assay (ELISA), a high titer antibody corresponds to an antibody present in a serum sample that remains positive in the assay after a dilution of the serum to approximately the range of 1:100-1:1000 in an appropriate dilution buffer, preferably, about 1:500. The preferred antigenic domains, however, have an epitope found on herpes simplex virus gG2 protein, hepatitis B virus s antigen (HBsAg), hepatitis B virus e antigen (HBeAg), hepatitis B virus c antigen (HBcAg), TT virus, and the poliovirus or combination thereof or comprise a sequence selected from the group consisting of SEQ. ID. Nos. 43-59.
The ligand/receptor specificity exchangers described herein can be made by conventional techniques in recombinant engineering and/or peptide chemistry. In some embodiments, the specificity and antigenic domains are made separately and are subsequently joined together (e.g., through linkers or by association with a common carrier molecule). In other embodiments, the specificity domain and antigenic domain are made as part of the same molecule. By one approach, a ligand/receptor specificity exchanger having a specificity domain joined to an antigenic domain is made by a peptide synthesizer. By another approach, a nucleic acid encoding the specificity domain fused to an antigenic domain is cloned into an expression construct, transfected to cells, and the ligand/receptor specificity exchanger is purified or isolated from the cells or cell supernatent.
Once the ligand/receptor specificity exchanger is made, it can be screened to determine its ability to interact with the receptor on the pathogen and/or an antibody specific for the antigenic domain. Thus, the term xe2x80x9ccharacterization assayxe2x80x9d is used to refer to an experiment or evaluation of the ability of a ligand/receptor specificity exchanger to interact with a receptor on a pathogen or cancer cell or fragment thereof and/or an antibody specific for the antigenic domain. Some characterization assays, for example, evaluate the ability of a ligand/receptor specificity exchanger to bind to a support having a receptor of a pathogen or fragment thereof disposed thereon or vice versa. Other characterization assays assess the ability of a ligand/receptor specificity exchanger to bind to an antibody specific for the antigenic domain of the ligand/receptor specificity exchanger. Still other characterization assays evaluate the ability of the ligand/receptor specificity exchanger to effect infection by the pathogen or cancer cell proliferation in cultured cell lines or diseased animals.
The ligand/receptor specificity exchangers described herein can be used as the active ingredients in pharmaceuticals for the treatment and prevention of pathogenic infection, as well as cancer, in animals including humans. The pharmaceutical embodiments can be formulated in many ways and may contain excipients, binders, emulsifiers, carriers, and other auxiliary agents in addition to the ligand/receptor specificity exchanger. Pharmaceuticals comprising a ligand/receptor specificity exchanger can be administered by several routes including, but not limited to, topical, transdermal, parenteral, gastrointestinal, transbronchial, and transalveolar. Ligand/receptor specificity exchangers can also be used as a coating for medical equipment and prosthetics to prevent infection or the spread of disease. The amount of ligand/receptor specificity exchanger provided in a pharmaceutical, therapeutic protocol, or applied to a medical device varies depending on the intended use, the patient, and the frequency of administration.
Some of the methods disclosed concern the administration of a ligand/receptor specificity exchanger to a subject in need of treatment and/or prevention of bacterial infection, fungal infection, viral infection, and cancer. By one approach, a subject suffering from bacterial infection is provided a ligand/receptor specificity exchanger that comprises a specificity domain, which interacts with a bacterial receptor. Similarly, a subject suffering from a viral infection can be provided a ligand/receptor specificity exchanger that comprises a specificity domain that interacts with a viral receptor and a subject suffering from cancer is provided a ligand/receptor specificity exchanger that comprises a specificity domain that interacts with a receptor on the cancer cells. Once a receptor/specificity exchanger complex is formed, it is contemplated that the pathogen or cancer cell is cleared from the body by complement fixation and/or macrophage degradation.
Methods of treatment and prevention of disease (e.g., bacterial, fungal, and viral infection, and cancer) are provided in which a subject suffering from disease or a subject at risk for contracting a disease is identified and then is provided a therapeutically effective amount of a ligand/receptor specificity exchanger that interacts with a receptor present on the etiological agent. Accordingly, subjects suffering from a bacterial infection, fungal infection, viral infection, or cancer are identified by conventional clinical and diagnostic evaluation and are provided a therapeutically effective amount of a ligand/receptor specificity exchanger that interacts with the particular pathogen or cancer cell. Although the ligand/receptor specificity exchangers described herein can be administered to all animals at risk of disease for prophylactic purposes, it may be desired to administer the ligand/receptor specificity exchangers only to those individuals that are in a high risk category (e.g., infants, the elderly, and those that come in close contact with pathogens). As stated above, high risk individuals are identified by currently available clinical and diagnostic techniques.
The section below provides more description of various types of ligand/receptor specificity exchangers that interact with receptors on bacteria, parasites, fungus, mold, viruses, and cancer cells.
The ligand/receptor specificity exchangers that interact with receptors on a pathogen have a variety of chemical structures but, in a general sense, they are characterized as having at least one region that binds to the receptor (the specificity domain) and at least one region that interacts with an antibody that is specific for an epitope of a pathogen or toxin (the antigenic domain). Preferred ligand/receptor specificity exchangers are peptides but some embodiments comprise derivatized or modified peptides or a peptidomimetic structure. For example, a typical peptide-based ligand/receptor specificity exchanger can be modified to have substituents not normally found on a peptide or to have substituents that are normally found on a peptide but are incorporated at regions that are not normal. In this vein, a peptide-based ligand/receptor specificity exchanger can be acetylated, acylated, or aminated and the substituents that can be included on the peptide so as to modify it include, but are not limited to, H, alkyl, aryl, alkenyl, alkynl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl or a 5 or 6 member aliphatic or aromatic ring. Thus, the term xe2x80x9cligand/receptor specificity exchangerxe2x80x9d is a broad one that encompasses modified or unmodified peptide structures, as well as peptidomimetics and chemical structures.
There are many ways to make a peptidomimetic that resembles a peptide-based ligand/receptor specificity exchanger. The naturally occurring amino acids employed in the biological production of peptides all have the L-configuration. Synthetic peptides can be prepared employing conventional synthetic methods, utilizing L-amino acids, D-amino acids, or various combinations of amino acids of the two different configurations. Synthetic compounds that mimic the conformation and desirable features of a peptide but that avoid the undesirable features, e.g., flexibility (loss of conformation) and bond breakdown are known as a xe2x80x9cpeptidomimeticsxe2x80x9d. (See, e.g., Spatola, A. F. Chemistry and Biochemistry of Amino Acids. Peptides, and Proteins (Weistein, B, Ed.), Vol. 7, pp. 267-357, Marcel Dekker, New York (1983), which describes the use of the methylenethio bioisostere [CH2S] as an amide replacement in erikephalin analogues; and Szelke et al., In peptides: Structure and Function, Proceedings of the Eighth American Peptide Symposium, (Hruby and Rich, Eds.); pp. 579-582, Pierce Chemical Co., Rockford, Ill. (1983), which describes renin inhibitors having both the methyleneamino [CH2 NH] and hydroxyethylene [CHOHCH2] bioisosteres at the Leu-Val amide bond in the 6-13 octapeptide derived from angiotensinogen, all of which are expressly incorporated by reference in their entireties).
In general, the design and synthesis of a peptidomimetic that resembles a ligand/receptor specificity exchanger involves starting with the sequence of the ligand/receptor specificity exchanger and conformation data (e.g., geometry data, such as bond lengths and angles) of a desired ligand/receptor specificity exchanger (e.g., the most probable simulated peptide), and using such data to determine the geometries that should be designed into the peptidomimetic. Numerous methods and techniques are known in the art for performing this step, any of which could be used. (See, e.g., Farmer, P. S., Drug Design, (Ariens, E. J. ed.), Vol. 10, pp. 119-143 (Academic Press, New York, London, Toronto, Sydney and San Francisco) (1980); Farmer, et al., in TIPS, 9/82, pp. 362-365; Verber et al., in TINS, 9/85, pp. 392-396; Kaltenbronn et al., in J. Med. Chem. 33: 838-845 (1990); and Spatola, A. F., in Chemistry and Biochemistry of Amino Acids. Peptides, and Proteins, Vol. 7, pp. 267-357, Chapter 5, xe2x80x9cPeptide Backbone Modifications: A Structure-Activity Analysis of Peptides Containing Amide Bond Surrogates. Conformational Constraints, and Relationsxe2x80x9d (B. Weisten, ed.; Marcell Dekker: New York, pub.) (1983); Kemp, D. S., xe2x80x9cPeptidomimetics and the Template Approach to Nucleation of xcex2-sheets and xcex1-helices in Peptides,xe2x80x9d Tibech, Vol. 8, pp. 249-255 (1990), all of which are expressly incorporated by reference in their entireties). Additional teachings can be found in U.S. Pat. Nos. 5,288,707; 5,552,534; 5,811,515; 5,817,626; 5,817,879; 5,821,231; and 5,874,529, all of which are expressly incorporated by reference in their entireties. Once the peptidomimetic is designed, it can be made using conventional techniques in peptide chemistry and/or organic chemistry.
Some embodiments comprise a plurality of specificity domains and/or a plurality of antigenic domains. One type of ligand/receptor specificity exchanger that has a plurality of specificity domains and/or antigenic domains is referred to as a xe2x80x9cmultimerized ligand/receptor specificity exchangerxe2x80x9d because it has multiple specificity domains and/or antigenic domains that appear in tandem on the same molecule. For example, a multimerized specificity domain may have two or more ligands that interact with one type of receptor, two or more ligands that interact with different types of receptors on the pathogen, and two or more ligands that interact with different types of receptors on different pathogens.
Similarly, a multimerized antigenic domain can be constructed to have multimers of the same epitope of a pathogen or different epitopes of a pathogen, which can also be multimerized. That is, some multimerized antigenic domains are multivalent because the same epitope is repeated. In contrast, some multimerized antigenic domains have more than one epitope present on the same molecule in tandem but the epitopes are different. In this respect, these antigenic domains are multimerized but not multivalent. Further, some multimerized antigenic domains are constructed to have different epitopes but the different epitopes are themselves multivalent because each type of epitope is repeated.
Some ligand/receptor specificity exchangers comprise other elements in addition to the specificity domain and antigenic domain such as sequences that facilitate purification, linkers that provide greater flexibility and reduce steric hindrance, and sequences that either provide greater stability to the ligand/receptor specificity exchanger (e.g., resistance to protease degradation) or promote degradation (e.g., protease recognition sites). For example, the ligand/receptor specificity exchangers can comprise cleavable signal sequences that promote cytoplasmic export of the peptide and/or cleavable sequence tags that facilitate purification on antibody columns, glutathione columns, and metal columns.
Ligand/receptor specificity exchangers can comprise elements that promote flexibility of the molecule, reduce steric hindrance, or allow the ligand/receptor specificity exchanger to be attached to a support or other molecule. These elements are collectively referred to as xe2x80x9clinkersxe2x80x9d. One type of linker that can be incorporated with a ligand/receptor specificity exchanger, for example, is avidin or streptavidin (or their ligandxe2x80x94biotin). Through a biotin-avidin/streptavidin linkage, multiple ligand/receptor specificity exchangers can be joined together (e.g., through a support, such as a resin, or directly) or individual specificity domains and antigenic domains can be joined. Another example of a linker that can be included in a ligand/receptor specificity exchanger is referred to as a xe2x80x9cxcex linkerxe2x80x9d because it has a sequence that is found on xcex phage. Preferred xcex sequences are those that correspond to the flexible arms of the phage. These sequences can be included in a ligand/receptor specificity exchanger (e.g., between the specificity domain and the antigenic domain or between multimers of the specificity and/or antigenic domains) so as to provide greater flexibility and reduce steric hindrance.
Additionally, ligand/receptor specificity exchangers can include sequences that either confer resistance to protease degradation or promote protease degradation. By incorporating multiple cysteines in a ligand/receptor specificity exchanger, for example, greater resistance to protease degradation can be obtained. These embodiments of the ligand/receptor specificity exchanger are expected to remain in the body for extended periods, which may be beneficial for some therapeutic applications. In contrast, ligand/receptor specificity exchangers can also include sequences that promote rapid degradation so as to promote rapid clearance from the body. Many sequences that serve as recognition sites for serine, cysteine, and aspartic proteases are known and can be included in a ligand/receptor specificity exchanger.
The section below describes the specificity domains in greater detail.
The types of specificity domains that can be used with a ligand/receptor specificity exchanger are diverse because a vast number of ligands are known to interact with receptors on bacteria, parasites, fungus, mold, viruses, and cancer cells. Many types of bacteria, parasites, fungus, mold, viruses, and cancer cells, for example, interact with extracellular matrix proteins. Thus, desired specificity domains comprise at least one ligand that has a peptide sequence that is present in an extracellular matrix protein. That is, a specificity domain can have a ligand that has a peptide sequence found in, for example, fibrinogen, collagen, vitronectin, laminin, plasminogen, thrombospondin, and fibronectin.
Investigators have mapped the regions of extracellular matrix proteins that interact with several receptors. (See e.g., McDevvit et al., Eur. J. Biochem., 247:416-424 (1997); Flock, Molecular Med. Today, 5:532 (1999); and Pei et al., Infect. and Immun. 67:4525 (1999), all of which are herein expressly incorporated by reference in their entirety). Some receptors bind to the same region of the extracellular matrix protein, some have overlapping binding domains, and some bind to different regions altogether. Preferably, the ligands that make up the specificity domain have an amino acid sequence that has been identified as being involved in adhesion to an extracellular matrix protein. It should be understood, however, that random fragments of known ligands for any receptor on a pathogen can be used to generate ligand/receptor specificity exchangers and these candidate ligand/receptor specificity exchangers can be screened in the characterization assays described infra to identify the molecules that interact with the receptors on the pathogen.
Some specificity domains have a ligand that interacts with a bacterial adhesion receptor including, but not limited to, extracellular fibrinogen binding protein (Efb), collagen binding protein, vitronectin binding protein, laminin binding protein, plasminogen binding protein, thrombospondin binding protein, clumping factor A (ClfA), clumping factor B (ClfB), fibronectin binding protein, coagulase, and extracellular adherence protein. Ligands that have an amino acid sequence corresponding to the C-terminal portion of the gamma-chain of fibrinogen have been shown to competitively inhibit binding of fibrinogen to ClfA, a Staphylococcus aureus adhesion receptor. (McDevvit et al., Eur. J. Biochem., 247:416-424 (1997)). Further, Staphylococcus organisms produce many more adhesion receptors such as Efb, which binds to the alpha chain fibrinogen, ClfB, which interacts with both the xcex1 and xcex2 chain of fibrinogen, and Fbe, which binds to the xcex2 chain of fibrinogen. (Pei et al., Infect. and Immun. 67:4525 (1999)). Accordingly, preferred specificity domains comprise at least 3 amino acids of a sequence present in a molecule (e.g., fibrinogen) that can bind to a bacterial adhesion receptor.
Specificity domains can also comprise a ligand that interacts with a viral receptor. Several viral receptors and corresponding ligands are known and these ligands or fragments thereof can be incorporated into a ligand/receptor specificity exchanger. For example, Tong et al., has identified an Hepadnavirus receptor, a 170 kd cell surface glycoprotein that interacts with the pre-S domain of the duck hepatitis B virus envelope protein (U.S. Pat. No. 5,929,220) and Maddon et al., has determined that the T cell surface protein CD4 (or the soluble form termedT4) interacts with gp120 of HIV (U.S. Pat. No. 6,093,539); both references are herein expressly incorporated by reference in their entireties. Thus, specificity domains that interact with a viral receptor can comprise regions of the pre-S domain of the duck hepatitis B virus envelope protein (e.g., amino acid residues 80-102 or 80-104) or regions of the T cell surface protein CD4 (or the soluble form termedT4) that interacts with gp120 of HIV (e.g., the extracellular domain of CD4/T4 or fragments thereof). Many more ligands for viral receptors exist and these molecules or fragments thereof can be used as a specificity domain.
Specificity domains can also comprise a ligand that interacts with a receptor present on a cancer cell. The proto-oncogene HER-2/neu (C-erbB2) encodes a surface growth factor receptor of the tyrosine kinase family, p185HER2. Twenty to thirty percent of breast cancer patients over express the gene encoding HER-2/neu (C-erbB2), via gene amplification. Thus, ligand/receptor specificity exchangers comprising a specificity domain that encodes a ligand for HER-2/neu (C-erbB2) are desirable embodiments. Many types of cancer cells also over express or differentially express integrin receptors. Many preferred embodiments comprise a specificity domain that interacts with an integrin receptor. Although integrins predominantly interact with extracellular matrix proteins, it is known that these receptors interact with other ligands such as invasins, RGD-containing peptides (i.e., Arginine-Glycine-Aspartate), and chemicals. (See e.g., U.S. Pat. Nos. 6,090,944 and 6,090,388; and Brett et al., Eur J. Immunol, 23:1608 (1993), all of which are hereby expressly incorporated by reference in their entireties). Ligands for integrin receptors include, but are not limited to, molecules that interact with a vitronectin receptor, a laminin receptor, a fibronectin receptor, a collagen receptor, a fibrinogen receptor, an xcex14xcex21 receptor, an xcex16xcex21 receptor, an xcex13xcex21 receptor, an xcex15xcex21 receptor, and an xcex1vxcex21 receptor. TABLE I also lists several preferred specificity domains. The specificity domains described above are provided for illustrative purposes only and in no way should be construed to limit the scope of specificity domains that can be used with the embodiments described herein.
The next section describes antigenic domains in greater detail.
The diversity of antigenic domains that can be used in the ligand/receptor specificity exchangers is also quite large because a pathogen or toxin can present many different epitopes. That is, the antigenic domains that can be incorporated into a ligand/receptor specificity exchanger include epitopes presented by bacteria, fungus, plants, mold, virus, cancer cells, and toxins. Desired antigenic domains comprise an epitope of a pathogen that already exists in a subject by virtue of naturally acquired immunity or vaccination. Epitopes of pathogens that cause childhood diseases, for example, can be used as antigenic domains.
Some embodiments have antigenic domains that interact with an antibody that has been administered to the subject. For example, an antibody that interacts with an antigenic domain on a specificity exchanger can be co-administered with the specificity exchanger. Further, an antibody that interacts with a ligand/receptor specificity exchanger may not normally exist in a subject but the subject has acquired the antibody by introduction of a biologic material (e.g., serum, blood, or tissue). For example, subjects that undergo blood transfusion acquire numerous antibodies, some of which can interact with an antigenic domain of a ligand/receptor specificity exchanger. Some preferred antigenic domains for use in a ligand/receptor specificity exchanger comprise viral epitopes including, but not limited to, the herpes simplex virus, hepatitis B virus, TT virus, and the poliovirus.
In some embodiments, it is also preferred that the antigenic domains comprise an epitope of a pathogen or toxin that is recognized by a xe2x80x9chigh-titer antibodyxe2x80x9d. Approaches to determine whether the epitope of a pathogen or toxin is recognizable by a high titer antibody are provided infra. Epitopes of a pathogen that can be included in an antigenic domain of a ligand/receptor specificity exchanger include epitopes on peptide sequences disclosed in Swedish Pat No. 9901601-6; U.S. Pat. No. 5,869,232; Mol. Immunol. 28: 719-726 (1991); and J. Med. Virol. 33:248-252 (1991); all references are herein expressly incorporated by reference in their entireties. TABLE II provides the amino acid sequence of several preferred antigenic domains.
The section following TABLE II, describes the preparation of ligand/receptor specificity exchangers in greater detail.
In some embodiments, the specificity and antigenic domains are made separately and are subsequently joined together (e.g., through linkers or by association with a common carrier molecule) and in other embodiments, the specificity domain and antigenic domain are made as part of the same molecule. For example, any of the specificity domains listed in TABLE I can be joined to any of the antigenic domains of TABLE II. Although the specificity and antigenic domains could be made separately and joined together through a linker or carrier molecule (e.g., a complex comprising a biotinylated specificity domain, streptavidin, and a biotinylated antigenic domain), it is preferred that the ligand/receptor specificity exchanger is made as a fusion protein. Thus, preferred embodiments include fusion proteins comprising any of the specificity domains listed in TABLE I joined to any of the antigenic domains of TABLE II.
Ligand/receptor specificity exchangers can be generated in accordance with conventional methods of protein engineering, protein chemistry, organic chemistry, and molecular biology. Additionally, some commercial enterprises manufacture made-to-order peptides and a ligand/receptor specificity exchanger can be obtained by providing such a company with the sequence of a desired ligand/receptor specificity exchanger and employing their service to manufacture the agent according to particular specifications (e.g., Bachem AG, Switzerland). Preferably, the ligand/receptor specificity exchangers are prepared by chemical synthesis methods (such as solid phase peptide synthesis) using techniques known in the art, such as those set forth by Merrifield et al., J. Am. Chem. Soc. 85:2149 (1964), Houghten et al., Proc. Natl. Acad. Sci. USA, 82:51:32 (1985), Stewart and Young (Solid phase peptide synthesis, Pierce Chem Co., Rockford, Ill. (1984), and Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman and Co., N.Y.; all references are herein expressly incorporated by reference in their entireties.
By one approach, solid phase peptide synthesis is performed using an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, Calif.). Each synthesis uses a p-methylbenzylhydrylamine solid phase support resin (Peptide International, Louisville, Ky.) yielding a carboxyl terminal amide when the peptides are cleaved off from the solid support by acid hydrolysis. Prior to use, the carboxyl terminal amide can be removed and the ligand/receptor specificity exchangers can be purified by high performance liquid chromatography (e.g., reverse phase high performance liquid chromatography (RP-HPLC) using a PepS-15 C18 column (Pharmacia, Uppsala, Sweden)) and sequenced on an Applied Biosystems 473A peptide sequencer. An alternative synthetic approach uses an automated peptide synthesizer (Syro, Multisyntech, Tubingen, Germany) and 9-fluorenylmethoxycarbonyl (fmoc) protected amino acids (Milligen, Bedford, Mass.).
While the ligand/receptor specificity exchangers can be chemically synthesized, it can be more efficient to produce these polypeptides by recombinant DNA technology using techniques well known in the art. Such methods can be used to construct expression vectors containing nucleotide sequences encoding a ligand/receptor specificity exchanger and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Alternatively, RNA capable of encoding a ligand/receptor specificity exchanger can be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in Oligonucleotide Synthesis, 1984, Gait, M. J. ed., IRL Press, Oxford, which is incorporated by reference herein in its entirety.
A variety of host-expression vector systems can be utilized to express the ligand/receptor specificity exchangers. Where the ligand/receptor specificity exchanger is a soluble molecule it can be recovered from the culture, i.e., from the host cell in cases where the peptide or polypeptide is not secreted, and from the culture media in cases where the peptide or polypeptide is secreted by the cells. However, the expression systems also encompass engineered host cells that express membrane bound ligand/receptor specificity exchangers. Purification or enrichment of the ligand/receptor specificity exchangers from such expression systems can be accomplished using appropriate detergents and lipid micelles and methods well known to those skilled in the art.
The expression systems that can be used include, but are not limited to, microorganisms such as bacteria (e.g., E. coli or B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing nucleotide sequences encoding a ligand/receptor specificity exchanger; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing nucleotide sequences encoding ligand/receptor specificity exchangers; insect cell systems infected with recombinant virus expression vectors (e.g., Baculovirus) containing nucleic acids encoding the ligand/receptor specificity exchangers; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing nucleic acids encoding ligand/receptor specificity exchangers.
In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the ligand/receptor specificity exchanger. For example, when a large quantity is desired (e.g., for the generation of pharmaceutical compositions of ligand/receptor specificity exchangers) vectors that direct the expression of high levels of fusion protein products that are readily purified can be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J., 2:1791 (1983), in which the ligand/receptor specificity exchanger coding sequence can be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye and Inouye, Nucleic Acids Res., 13:3101-3109 (1985); Van Heeke and Schuster, J. Biol. Chem., 264:5503-5509 (1989)); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The ligand/receptor specificity exchanger gene coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of ligand/receptor specificity exchanger gene coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus, (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (E.g., see Smith et al., J. Virol. 46: 584 (1983); and Smith, U.S. Pat. No. 4,215,051).
In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, a nucleic acid sequence encoding a ligand/receptor specificity exchanger can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the ligand/receptor specificity exchanger gene product in infected hosts. (See e.g., Logan and Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659 (1984)). Specific initiation signals can also be required for efficient translation of inserted ligand/receptor specificity exchanger nucleotide sequences (e.g., the ATG initiation codon and adjacent sequences). In most cases, an exogenous translational control signal, including, perhaps, the ATG initiation codon, should be provided. Furthermore, the initiation codon should be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can also be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bittner et al., Methods in Enzymol., 153:516-544 (1987)).
In addition, a host cell strain can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for some embodiments. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the ligand/receptor specificity exchangers described above can be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells are allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn are cloned and expanded into cell lines. This method is advantageously used to engineer cell lines which express a ligand/receptor specificity exchanger.
A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and adenine phosphoribosyltransferase (Lowy, et al., Cell 22:817 (1980)) genes can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al., Proc. Natl. Acad. Sci. USA 77:3567 (1980)); O""Hare, et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., J. Mol. Biol. 150:1 (1981)); and hygro, which confers resistance to hygromycin (Santerre, et al., Gene 30:147 (1984)).
The following section describes the ligand/receptor specificity exchanger characterization assays in greater detail.
Preferably, ligand/receptor specificity exchangers are analyzed for their ability to interact with a receptor and/or the ability to interact with an antibody that may be present in a subject. The term xe2x80x9ccharacterization assayxe2x80x9d refers to an assay, experiment, or analysis made on a ligand/receptor specificity exchanger, which evaluates the ability of a ligand/receptor specificity exchanger to interact with a receptor (e.g., a surface receptor present in bacteria, virus, mold, or fungi) or an antibody (e.g., an antibody that recognizes an epitope found on a pathogen), or effect the proliferation of a pathogen. Encompassed by the term xe2x80x9ccharacterization assayxe2x80x9d are binding studies (e.g., enzyme immunoassays (EIA), enzyme-linked immunoassays (ELISA), competitive binding assays, computer generated binding assays, support bound binding studies, and one and two hybrid systems), and infectivity studies (e.g., reduction of viral infection, propagation, and attachment to a host cell).
Preferred binding assays use multimeric agents. One form of multimeric agent concerns a composition comprising a ligand/receptor specificity exchanger, or fragments thereof disposed on a support. Another form of multimeric agent involves a composition comprising a receptor or an antibody specific for the antigenic domain of a ligand/receptor specificity exchanger disposed on a support. A xe2x80x9csupportxe2x80x9d can be a carrier, a protein, a resin, a cell membrane, or any macromolecular structure used to join or immobilize such molecules. Solid supports include, but are not limited to, the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, animal cells, Duracyte(copyright), artificial cells, and others. A ligand/receptor specificity exchanger can also be joined to inorganic supports, such as silicon oxide material (e.g. silica gel, zeolite, diatomaceous earth or aminated glass) by, for example, a covalent linkage through a hydroxy, carboxy, or amino group and a reactive group on the support.
In some multimeric agents, the macromolecular support has a hydrophobic surface that interacts with a portion of the ligand/receptor specificity exchanger, receptor or ligand by a hydrophobic non-covalent interaction. In some cases, the hydrophobic surface of the support is a polymer such as plastic or any other polymer in which hydrophobic groups have been linked such as polystyrene, polyethylene or polyvinyl. Additionally, a ligand/receptor specificity exchanger, receptor or an antibody specific for the antigenic domain of a ligand/receptor specificity exchanger can be covalently bound to supports including proteins and oligo/polysaccarides (e.g. cellulose, starch, glycogen, chitosane or aminated sepharose). In these later multimeric agents, a reactive group on the molecule, such as a hydroxy or an amino group, is used to join to a reactive group on the carrier so as to create the covalent bond. Additional multimeric agents comprise a support that has other reactive groups that are chemically activated so as to attach the ligand/receptor specificity exchanger, receptor, or antibody specific for the antigenic domain of a ligand/receptor specificity exchanger. For example, cyanogen bromide activated matrices, epoxy activated matrices, thio and thiopropyl gels, nitrophenyl chloroformate and N-hydroxy succinimide chlorformate linkages, or oxirane acrylic supports can be used. (Sigma). Furthermore, in some embodiments, a liposome or lipid bilayer (natural or synthetic) is contemplated as a support and a ligand/receptor specificity exchanger, receptor, or an antibody specific for the antigenic domain of a ligand/receptor specificity exchanger can be attached to the membrane surface or are incorporated into the membrane by techniques in liposome engineering. By one approach, liposome multimeric supports comprise a ligand/receptor specificity exchanger, receptor, or an antibody specific for the antigenic domain of a ligand/receptor specificity exchanger that is exposed on the surface.
The insertion of linkers (e.g., xe2x80x9cxcex linkersxe2x80x9d engineered to resemble the flexible regions of xcex phage) of an appropriate length between the ligand/receptor specificity exchanger, receptor, or antibody specific for the antigenic domain of a ligand/receptor specificity exchanger and the support are also contemplated so as to encourage greater flexibility and overcome any steric hindrance that can be presented by the support. The determination of an appropriate length of linker that allows for optimal binding can be found by screening the attached molecule with varying linkers in the characterization assays detailed herein.
Several approaches to characterize ligand/receptor specificity exchangers employ a multimeric described above. For example, support-bound ligand/receptor specificity exchanger can be contacted with xe2x80x9cfreexe2x80x9d adhesion receptors and an association can be determined directly (e.g., by using labeled adhesion receptors) or indirectly (e.g., by using a labeled ligand directed to an adhesion receptor). Thus, candidate ligand/receptor specificity exchangers are identified as bona fide ligand/receptor specificity exchangers by virtue of the association of the receptors with the support-bound candidate ligand/receptor specificity exchanger. Alternatively, support-bound adhesion receptors can be contacted with xe2x80x9cfreexe2x80x9d ligand/receptor specificity exchangers and the amount of associated ligand/receptor specificity exchanger can be determined directly (e.g., by using labeled ligand/receptor specificity exchanger) or indirectly (e.g., by using a labeled antibody directed to the antigenic domain of the ligand/receptor specificity exchanger). Similarly, by using an antibody specific for the antigenic domain of a ligand/receptor specificity exchanger disposed on a support and labeled ligand/receptor specificity exchanger (or a secondary detection reagent, e.g., a labeled receptor or antibody to the ligand/receptor specificity exchanger) the ability of the antibody to bind to the antigenic domain of the ligand/receptor specificity exchanger can be determined.
Some characterization assays evaluate the ability of the ligand/receptor specificity exchanger to interact with the target receptor and the redirecting antibody while other characterization assays are designed to determine whether a ligand/receptor specificity exchanger can bind to both the target receptor and the redirecting antibody. In general, the characterization assays can be classified as: (1) in vitro characterization assays, (2) cellular characterization assays, and (3) in vivo characterization assays.
A discussion of each type of characterization assay is provided in the following sections.
There are many types of in vitro assays that can be used to determine whether a ligand/receptor specificity exchanger binds to a particular receptor and whether an antibody found in a subject can bind to the ligand/receptor specificity exchanger. Most simply, the receptor is bound to a support (e.g., a petri dish) and the association of the ligand/receptor specificity exchanger with the receptor is monitored directly or indirectly, as described above. Similarly, a primary antibody directed to the antigenic domain of a ligand/receptor specificity exchanger (e.g., an antibody found in a subject) can be bound to a support and the association of a ligand/receptor specificity exchanger with the primary antibody can be determined directly (e.g., using labeled ligandireceptor specificity exchanger) or indirectly (e.g., using labeled receptor or a labeled secondary antibody that interacts with an epitope on the ligand/receptor specificity exchanger that does not compete with the epitope recognized by the primary antibody).
Another approach involves a sandwich-type assay, wherein the receptor is bound to a support, the ligand/receptor specificity exchanger is bound to the receptor, and the primary antibody is bound to the ligand/receptor specificity exchanger. If labeled primary antibody is used, the presence of a receptor/specificity exchanger/primary antibody complex can be directly determined. The presence of the receptor/specificity exchanger/primary antibody complex can also be determined indirectly by using, for example, a labeled secondary antibody that reacts with the primary antibody at an epitope that does not interfere with the binding of the primary antibody to the ligand/receptor specificity exchanger. In some cases, it may be desired to use a labeled tertiary antibody to react with an unlabeled secondary antibody, thus, forming a receptor/specificity exchanger/primary antibody/secondary antibody/labeled tertiary antibody complex.